A critical role of plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase in the control of plant metabolism and development

Abstract

Glycolysis is a central metabolic pathway that provides energy and generates precursors for the synthesis of primary metabolites such as amino acids and fatty acids.^1–3 In plants, glycolysis occurs in the cytosol and plastids, which complicates the understanding of this essential process.¹ As a result, the contribution of each glycolytic pathway to the specific primary metabolite production and the degree of integration of both pathways is still unresolved. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described biochemically. But, up to now, little attention had been paid to GAPCps, probably because they have been considered as “minor isoforms” that catalyze a reversible reaction in plastids where it has been assumed that key glycolytic intermediates are in equilibrium with the cytosol. In the associated study,⁴ we have elucidated the crucial role of Arabidopsis GAPCps in the control of primary metabolism in plants. GAPCps deficiency affects amino acid and sugar metabolism and impairs plant development. Specifically, GAPCp deficiency affects the serine supply to roots, provoking a drastic phenotype of arrested root development. Also, we show that the phosphorylated serine biosynthesis pathway is critical to supply serine to non-photosynthetic organs such as roots. These studies provide new insights of the contribution of plastidial glycolysis to plant metabolism and evidence the complex interactions existing between metabolism and development.

GAPCps are “Minor” GAPDH Isoforms Located in the Chloroplast/Plastids

Although enzymes of the glycolytic pathway have been detected in plastids,⁵ the existence of a full active set of these enzymes in chloroplasts has been controversial.⁶ In the Arabidopsis genome there are described four glycolytic GAPDH phosphorylating isoforms (GAPC1, GAPC2, GAPCp1 and GAPCp2). Two of them (GAPCps) contain a putative plastid localization sequence. By expressing GAPCp-GFP fusion proteins in Arabidopsis, we demonstrated that GAPCp1 and GAPCp2 can be localized in both chloroplasts and plastids. We detected transcripts of GAPCp1 and GAPCp2 in green and non green organs although they were more abundant in roots and flowers. Both GAPCp1 and GAPCp2 showed a very low gene expression level and activity as compared to other GAPDH isoforms. Nevertheless, we thought that the strategic situation of GAPCps, in the middle of the glycolytic pathway, could make them important players in the control of primary metabolism.

GAPCp Deficiency Leads to Root Developmental Arrest and Impairs the C/N Balance

Because it is difficult to define the contribution of each glycolytic enzyme and pathway to the primary metabolism of plant cells by only biochemical approaches, our group followed a gain- and loss-of-function approach in Arabidopsis. We generated several gapcp double mutant lines (gapcp1gapcp2) that showed a much more drastic phenotype than that observed in other lines with reduced activity of cytosolic GAPDHs.^7,8 The most evident developmental defect in gapcp1gapcp2 was the arrest of the primary root growth which indicated that GAPCp is essential for the development of specific plant organs. These developmental alterations observed in the mutants were accompanied by impairment of the sugar and amino acid metabolism. The starch and total soluble sugar contents in gapcp1gapcp2 were increased by more than 80% as compared to wild type plants. The expression of genes and activities of enzymes involved in starch and sucrose biosynthesis such as sucrose-phosphate-synthase, starch synthase, starch phosphorylase, ADP-glucose pyrophosphorylase and sucrose synthase were also upregulated in gapc1gapcp2. This indicated that the starch-excess phenotype of gapc1gapcp2 is ascribed to activation of the starch biosynthetic machinery rather than to incapacity to metabolize it.

As the plastidial glycolytic pathway also provides precursors for several amino acid biosynthesis, we measured free amino acid content in gapcp1gapcp2. We found that the total free amino acids in the mutant roots were increased as compared to wild type plants. However, mutants had a serine deficiency in their roots. This was consistent with a possible function of GAPCp since this enzyme along with the phosphoglycerate kinase synthesizes 3-phosphoglycerate (3-PGA) which is the precursor of serine biosynthesis in the plastids through the so called phosphorylated pathway.

Serine Rescues the Root Developmental Arrest and All the Metabolic Changes Measured in gapcp1gapcp2

The severe primary root growth arrest caused by GAPCp loss-of-function was rescued by serine supplementation to the growth medium. In addition, serine supply restored normal starch and sucrose biosynthetic enzymes activities and normal levels of carbohydrates in roots and the aerial part of gapcp1gapcp2. These results indicated that the major factor affecting gapcp1gapcp2 metabolism and development was serine deficiency and provide in vivo evidence that the main function of GAPCp in roots is to supply 3-PGA for serine biosynthesis. We proposed that GAPCp deficiency in gapcp1gapcp2 is limiting the 3-PGA availability to the plastidial phosphorylated pathway of serine biosynthesis. Then, 3-PGA provided from other sources to gapcp1gapcp2 root plastids, such as that transported from the cytosol or that obtained from phosphoenol-pyruvate through the reactions catalyzed by phosphoglycerate mutase and enolase, should not be enough to compensate the 3-PGA not produced by GAPCp disruption. According to this hypothesis, the 3-PGA provided by GAPCp and phosphoglycerate kinase activity seems to be the main source of this metabolite in the root plastids.

The plastidic and cytosolic glycolysis interact through highly selective transporters present in the inner plastid envelope⁹ which may suggests that key glycolytic intermediates are fully equilibrated in both compartments. Our results indicate that this is not the case, at least for some glycolytic intermediates and/or at some developmental stages. So this idea may need to be re-examined.

Does Serine Only have a Metabolic Function in Plants?

Although the relative serine content in roots of gapcp1gapcp2 was decreased by about 50% as compared to wild type plants, the absolute content was only reduced by 17%. The next question that arose in the light of these data is whether the profound developmental alterations observed in gapcp1gapcp2 are only due to a metabolic function of serine or if a specific role of this amino acid and/or its derivatives in root development should also be considered. Serine is involved in the first committed step of the sphingolipid biosynthesis, a reaction catalyzed by serine palmitoyltransferase. The gapcp1gapcp2 shares some phenotypes with sphingolipid biosynthetic mutants, especially the severe reduction of root growth.¹⁰ Dwarfing was the most marked phenotype associated with the partial suppression of serine palmitoyltransferase LCB1 gene in RNAi Arabidopsis plants.¹¹ These authors concluded that the small size of plants appeared to be attributable to reduced cell expansion as we have also shown for gapcp1gapcp2. Then, one of the important physiological functions rescued by serine supply could be the recovery of the sphingolipid content.

In the last years, much attention has been paid to the transcriptional and translational control of plant development. Recently, our group and others have shown how small metabolic changes can have drastic effects on development. The question that now arises in the field of plant development is: what comes first, the metabolic chicken or the transcriptional/translational egg?

Conclusions

Knowledge about plastidic glycolysis is scarce and mainly based on biochemical evidence. We have described for the first time in plants the in vivo functional characterization of GAPCps and their interaction with the serine biosynthesis pathways. Our results point out the importance of plastidial glycolytic enzymes in plant primary metabolism, specifically of GAPCps, and suggest that compartmentalization of glycolysis between cytosol and plastids is functionally significant.

Addendum to: Muñoz-Bertomeu J, Cascales-Miñana B, Mulet JM, Baroja-Fernàndez E, Pozueta-Romero J, Kuhn JM, et al. Plastidial glyceraldehyde-3-phosphate dehydrogenase deficiency leads to altered root development and affects the sugar and amino acid balance in Arabidopsis. Plant Physiol. 2009;151:541–558. doi: 10.1104/pp.109.143701.